The involvement of the double-stranded RNA-activated proteinkinase PKR in the regulation of the myogenic process was investigated.For this purpose, the murine myogenic cell line C2C12 was used.The cells were first cultivated in either growth medium or differentiationmedium (DM), and the activation of PKR during differentiationwas determined by monitoring its enzymatic activity and by immunoblotanalysis. A significant increase in both parameters was detectedalready at 24 h in DM, whereas in cells grown in growth medium,the increase was evident only after 96 h, when spontaneous differentiationwas observed in highly crowded cultures. Consequently, we establishedthe direct effect of PKR activation on the myogenic process.C2C12 cells were transfected with an expression vector harboringa cDNA molecule encoding human PKR fused to the inducible metallothioneinpromoter. One of the clones (clone 8) expressing high levelsof PKR was selected and further analyzed. In the presence ofZnCl2, which activates the promoter, the rate of cell growthof the transfected cells was clearly reduced compared to thatof wild-type C2C12 cells transfected with only the neomycin-resistantgene (C2-NEO). In addition, altered morphology with partialfusion was observed. Biochemically, an increase in creatinekinase activity accompanied by an increased rate of expressionof the myogenic protein troponin T and the myogenic transcriptionfactors myoD and myogenin was detected in clone 8 cells exposedto ZnCl2. Most importantly, an induction in the level of cyclin-dependentkinase inhibitor p21WAF1 and an increase in the level of theunderphosphorylated active form of the tumor suppressor proteinpRb concomitant with the down-regulation of cyclin D1 and c-mycwere also evident in the transfected clones. These changes weresimilar to those observed in normal C2C12 cells cultivated inDM. We conclude that PKR is an important regulatory proteinparticipating in the myogenic process.

PKR, a double-stranded RNA-activated serine-threonine proteinkinase, has been originally described as an IFN-inducible enzymeimplicated in antiviral activity (1, 2)
. Upon activation,the enzyme molecule is first autophosphorylated at several sites,followed by the phosphorylation of the target molecules (3)
.Several such targets have been reported. The best characterizedof these is the subunit of the translation initiation factoreIF-2. The factor is phosphorylated on serine residue 51; consequently,the exchange of the eIF-2-bound GDP with GTP by exchange factoreIF-2B is blocked (4)
, resulting in the inhibition of proteinsynthesis. In addition, the transcription factor inhibitor IB(5)
and the HIV-specific Tar-binding protein tat (6)
werealso described as possible (although not necessarily direct)targets phosphorylated by PKR. The genes encoding PKR both fromhuman (7)
and mouse (8)
origin were isolated and characterized,and the structural domains present in the protein molecule wereelucidated. The NH2-terminal portion of the enzyme appears tocontain the double-stranded RNA binding motif and the abilityto interact with other protein molecules, including itself (9,10, 11)
. However, it is still not clear whether dimerizationis indeed required for the PKR-mediated biological effects (12)
.The catalytic domains of the enzyme, on the other hand, areall located in the COOH-terminal region (13)
. As can be judgedfrom its mode of action, PKR is not involved only in antiviralactivity but has a much broader biological significance. Ithas been clearly demonstrated that ectopic expression of negativedominant mutants of PKR in NIH/3T3 mouse fibroblasts resultedin their malignant transformation (14, 15, 16)
, indicatingthat the enzyme has a tumor-suppressive effect and is most likelyassociated with the regulation of cell growth. In addition,overexpression of wild-type PKR was reported to induce apoptosisin susceptible cells, whereas expression of bcl-2 or mutatedPKR was able to protect the cells from this event (17, 18)
.Interestingly, PKR can modulate the function of the signal transducerand activator of transcription STAT1 by association or dissociationbetween these two proteins (19)
. Finally, PKR was reportedto down-regulate the expression of c-myc in growth-retardedM1 cells transfected with wild-type PKR (20)
. Taken together,these results suggest that the enzyme fulfills a pivotal regulatoryrole within the cell. However, it is still not clear whetherit is involved in differentiation processes. To address thisquestion, we studied the effect of the ectopic expression ofPKR on myogenesis.

Skeletal muscle cell cultures are an excellent experimentaltool for the study of differentiation in vitro. Upon withdrawalfrom the cell cycle, myogenic committed cells known as myoblastsfuse to form myotubes. This process is well controlled by aseries of myogenic specific transcription factors, the myoDfamily. The family consists of myoD, myogenin, Myf-5, and MRF4,all of which contain a DNA-binding basic region and a helix-loop-helixmotif responsible for interaction with other proteins (21)
,mostly with products of the E2A gene, such as E12 or E47 (22)
.This complex binds to an element on the DNA termed the E boxwith the consensus sequence CANNTG. Although myoD homodimersare as stable as the myoD-E12 heterodimers, only the latterbind to the E box (23)
. It should be noted, however, that additionalproteins are involved in modulating the activity of the myoDfamily (24)
.

IFN3
has been previously shown to induce morphological andbiochemical changes in several cell systems, including skeletalmyogenic cell cultures (25, 26, 27)
. In addition, the expressionof both PKR and 2-5A synthetase, another IFN-induced protein,was reported to increase during myogenic differentiation invitro(28)
. Furthermore, various agents that inhibit myogenesiswere also effective in interfering with the expression of theseproteins (29, 30)
. Most recently, it has been shown that anadditional IFN-inducible protein, p202, increased during skeletalmuscle differentiation (31)
. However, these elevated levelsmay be fortuitous, and more direct evidence is needed to clarifywhether PKR plays a role in the myogenic process. In this report,we show that ectopic expression of PKR in myogenic cells resultsin morphological, molecular, and biochemical alterations associatedwith skeletal muscle differentiation.

Activation of PKR during Myogenic Differentiation of C2C12 Cells.
We had to establish first whether PKR is specifically activatedduring induction of differentiation of the myogenic cell lineC2C12. The cells were cultivated in either GM or DM. Cell extractswere prepared at different times, and the presence of PKR wasidentified by immunoblot analysis using polyclonal antibodiesdirected against human PKR. The PKR protein appeared as a broadband of 6668 kDa (Fig. 1A)
. In parallel, the level ofthe biologically active protein was determined by monitoringits enzymatic activity under the same conditions. The 32P-labeledautophosphorylated form of PKR, which was 67 kDa in size, wasan indication of enzymatic activity (Fig. 1B)
. The basal levelof the PKR protein at the initiation of the experiment (zerotime) was low (data not shown) and did not change within thefirst 24 h in GM (Fig. 1A
, DIVISION, lane 1; Fig. 1C
). However,a gradual increase was observed with time in culture up to 144h (Fig. 1 A
, C
, DIVISION). It should be noted that a few myotubeswere always visible in crowded C2C12 cells even when cultivatedin GM, an indication of spontaneous differentiation. The kineticsof PKR enzymatic activity obtained with C2C12 dividing cells(Figs. 1, B
and D
, DIVISION) was similar to that observed withthe level of the PKR protein, demonstrating that the gradualincrease in this activity reflects an increase in the totalamount of PKR. In contrast, in the case of C2C12 cells grownin DM, a significant increase in both the level of PKR and itsenzymatic activity was already evident at 24 h after the mediumchange (Fig. 1, A
and B
, DIFFERENTIATION). The level of bothparameters remained constant up to 96 h and was followed bya decrease. At this time, the cultures were fully differentiated.

Fig. 1. Activation of PKR dur-ing myogenic differentiation of C2C12 cells. The cells were seeded in GM, and the medium was replaced with either GM (DIVISION) or DM (DIFFERENTIATION) 24 h later. At the indicated times thereafter, cell extracts were prepared, and PKR was identified either by immunoblot analysis (A) or by its enzymatic activity (B). Densitometry of A and B is presented in C and D, respectively.

Transfection of PKR into C2C12 Cells.
The fact that PKR is induced during differentiation of C2C12cells does not necessarily imply that the enzyme plays a rolein myogenesis. To show its direct involvement in the process,we constructed plasmid pMPKR, which harbors a cDNA encodinghuman PKR fused to the metallothionion promoter (see "Materialsand Methods"). This plasmid was cotransfected into C2C12 cellswith pSVneo, and 25 neomycin-resistant clones were isolatedand expanded. Eight clones expressed high levels of PKR in responseto the presence of ZnCl2 (which activates the promoter). Theresults obtained with a representative clone, clone 8, are presentedin Fig. 2
; however two additional clones that were analyzedsimilarly yielded comparable results that were not includedfor the sake of simplicity. For control cells, we used a clonetransfected with pSVneo only. This clone was designated C2-NEO.Immunoblot analysis as well as a determination of PKR enzymaticactivity indicated that the basal level of PKR in either C2-NEOor clone 8 cells was rather low; however, only clone 8 cellsexposed to ZnCl2 for 24 h responded with an elevated level ofPKR protein (Fig. 2A
, lane 6) and an increase in its enzymaticactivity (Fig. 2B
, lane 6). In contrast, both C2-NEO and clone8 cells responded equally well to a 24-h treatment with IFN(Fig. 2, A
and B
, lanes 2 and 5), an indication that the endogenousPKR-encoding gene is functional in these types of cells.

Fig. 2. Expression of PKR in transfected clone 8 cells. C2-NEO and clone 8 cells were treated with either IFN or ZnCl2 (Zn2+) for 24 h. One group of cultures remained untreated (C). Cell extracts were prepared, and PKR was identified either by immunoblot analysis (A) or by enzymatic activity (B). Densitometry of A and B is presented in C and D, respectively.

Morphological Alterations and Growth Characteristics of PKR-expressing C2C12 Cells.
To establish whether PKR is indeed involved in the initiationof the myogenic process, it was important to study the effectof its ectopic expression on a variety of biological parameters.First, C2-NEO and clone 8 cells were grown in GM in the presenceor absence of ZnCl2 for 96 h, and changes in cell morphologywere examined microscopically. Exposure of control cells toZnCl2 did not result in any alterations in the morphology ordensity of treated cells compared to those of untreated cells(compare Fig. 3, A
and C
). On the other hand, a striking differencewas observed when clone 8 cells were similarly analyzed. Celldensity was greatly reduced in ZnCl2-treated cultures, mostof the cells developed extended processes, and about 30% formedshort myotubes comprised of two to three cells (compare Fig.3, B
and D
). The retarded growth rate of clone 8 cells exposedto ZnCl2 was confirmed in the experiments described in
Fig.5
. The growth rates of normal C2C12 cells grown in GM or DMwere compared. Vital cell counts were performed daily afterthe medium change. Cells were labeled in parallel for 1.5 hwith [3H]thymidine, and the rate of DNA synthesis was determined.As expected, both parameters were significantly reduced in differentiatingversus dividing C2C12 cells (Fig. 4, A
and B
). We then comparedthe rates of growth and DNA synthesis of C2-NEO and clone 8cells cultivated in GM in the presence or absence of ZnCl2.It is clearly shown that whereas C2-NEO cells were hardly affectedby exposure to ZnCl2, the growth rate (Fig. 4C
) and kineticsof DNA synthesis (Fig. 4D
) were decreased in clone 8 cellstreated with ZnCl2. We conclude that cultivation in DM and ectopicexpression of PKR induce similar effects on the growth characteristicsof C2C12 cells.

Fig. 3. Effect of ZnCl2 on the morphology of C2C12 cell variants. C2-NEO (A and C) and clone 8 (B and D) cells were seeded in GM (-Zn2+) (A and B). Some cultures were exposed to ZnCl2 3 h later (+Zn2+) (C and D). After an additional 96 h, the cell morphology was examined with a phase-contrast microscope (x125).

Fig. 4. Kinetics of cell growth and DNA synthesis. C2C12 cells were cultured in GM, and after 24 h, the medium was replaced with either GM (DIVISION) or DM (DIFFERENTIATION). At the indicated times thereafter, viable cell counts were performed (A). The cultures were labeled in parallel with [3H]-thymidine, and acid-insoluble radioactivity was determined (B). Similarly, C2-NEO and clone 8 cells were seeded in GM (Ct). Some of the cultures were treated with ZnCl2 3 h later (Zn). The rates of cell growth (C) and DNA synthesis (D) were determined as described above.

Fig. 5. Determination of muscle-specific proteins. C2C12 cells were grown in GM (DIVISION) or DM (DIFFERENTIATION). Creatine kinase activity was determined at the indicated times by an enzymatic assay (A, I), and troponin T was identified by immunoblot analysis (B, I). In parallel, C2-NEO (A, II and B, II) and clone 8 cells (A, II; B, III) cultivated in GM in the absence (control, C) or presence of ZnCl2 were similarly analyzed.

Appearance of Muscle-specific Proteins and Transcription Factors in C2C12 Transfected Cells.
The reduced rate of proliferation of muscle cultures is accompaniedby the expression of muscle-specific proteins. This was confirmedin our study by the following observation. The levels of creatinekinase activity and troponin T gradually increased with timeup to 120 h when C2C12 cells were grown in DM. In contrast,when cells were cultivated in GM, the level of these muscle-specificproteins remained low, and an increase became evident only after96144 h (Fig. 5
, A
, I
and B
, I
). At this late time,spontaneous differentiation was observed in highly crowded cultures.We next determined whether the growth inhibition detected inclone 8 cells expressing PKR is also accompanied by an elevatedlevel of muscle-specific proteins. C2-NEO and clone 8 cellswere grown in the presence and absence of ZnCl2, and the levelsof both creatine kinase activity and troponin T were determinedat daily intervals. The results demonstrate that the presenceof ZnCl2 did not affect the level of either protein in C2-NEOcells (Fig. 5
, A
, II
and B
, II
). However, the appearanceof both proteins was significantly accelerated in ZnCl2-treatedversus untreated clone 8 cells (Fig. 5
, A
, II
and B
, III
),indicating that the ectopic expression of PKR induces the synthesisof muscle-specific proteins.

Next, we wanted to provide evidence that the myogenic transcriptionfactor myoD (32)
is also expressed in clone 8 cells expressingPKR. Total RNA was therefore extracted from ZnCl2-treated anduntreated clone 8 cultures, and Northern blot analysis was performedusing a myoD-specific probe. For comparison, a similar analysiswas performed on dividing and differentiating C2C12 cells. Theresults demonstrate that whereas a significant increase in thelevel of myoD-specific RNA transcripts was observed in C2C12cells grown in GM only after 120 h (Fig. 6
, A
, I)
, a majorincrease was detected in differentiating cells cultivated for24 h in DM, followed by a decrease only at 144 h (Fig. 6
, A
,II)
. Thus, as expected, differentiation of C2C12 cells is accompaniedby an increased expression of the myoD-encoding gene. In parallel,it is clearly shown that in ZnCl2-treated clone 8 cells (ectopicallyexpressing PKR), an elevated expression of myoD was observedat 72120 h (Fig. 6
, B
, II)
, whereas in control (untreated)cells, as in the case of dividing C2C12 cells, an increasedexpression of myoD was detected only at 120 h (Fig. 6
, B
,I)
. These results were confirmed by immunoblot analysis inwhich the level of the myoD protein as well as that of myogenin,a second myogenic transcription factor, was established. Theresults indicate that the amount of both proteins increasedin differentiating C2C12 cells, with a peak observed at 48 hin DM, followed by a decrease thereafter. In dividing cells,on the other hand, the amount remained low, and an increasewas observed only at 120144 h (Fig. 7
, A
, I
and B
,I
). We then performed a similar analysis on clone 8 cells grownin GM in the presence or absence of ZnCl2, using C2-NEO cellsgrown under similar conditions as an additional control. Asexpected, no effect of ZnCl2 was observed on the level of myogeninor myoD in C2-NEO cells (Fig. 7
, A
, II
and B
, II
). However,in the case of clone 8 cells, the level of both proteins increasedat least 24 h earlier in ZnCl2-treated cells compared to untreatedcells (Fig. 7
, A
, III
and B
, III)
. We conclude that activationof PKR in transfected cells enhances the synthesis of myogenictranscription factors.

Fig. 6. Detection of myoD-specific RNA transcripts. Total RNA was extracted from C2C12 cells grown in GM (A, I) or DM (A, II) and from untreated (B, I) or ZnCl2-treated (B, II) clone 8 cells. The amount of RNA in individual samples is shown in the ethidium bromide-stained gels presented in each section. Northern blot analysis was performed using a myoD-specific probe.

Fig. 7. Determination of myogenic transcription factors. C2C12 (I), C2-NEO (II), and clone 8 (III) cells were grown in the appropriate medium indicated in the figure, as described in Fig. 5. The levels of myogenin (A) and myoD (B) were determined by immunoblot analysis.

Effect of Ectopic Expression of PKR on Cell Cycle-associated Proteins.
Myogenic cells withdraw from the cell cycle during terminaldifferentiation. This process is associated with changes inthe expression of cell cycle-regulated proteins (for a review,see Walsh and Perlman, Ref. 33
). To explore whether ectopicexpression of PKR is accompanied by some of these changes, westudied the kinetics of c-myc, cyclin D1, the CDK inhibitorp21WAF1, and pRb synthesis, also known to be affected by IFN(34, 35)
. In agreement with earlier reports (33)
, the inductionof p21WAF1 synthesis, the down-regulation of cyclin D1, andthe accumulation of the underphosphorylated form of pRb wereobserved in C2C12 differentiating cells after 24 h in DM. Individing cells cultivated in GM, these changes occurred onlyat 120 h (Fig. 8
, ABC
, I
). In a similar analysis performedon C2-NEO cells grown in the presence or absence of ZnCl2, acomparable pattern of the expression of p21WAF1, cyclin D1,or pRb was evident in these two cell populations (Fig. 8
, A
B
C
,II
). However, in ZnCl2-treated clone 8 cells, we observed anincreased synthesis of p21WAF1, as indicated by a 3.5- and 5.3-foldincrease over the background level at 72 and 96 h after exposureto ZnCl2, respectively (Fig. 8
, A
, III)
. This is in contrastto a 1.3- and 1.2-fold increase in the level of p21WAF1 detectedat 72 and 96 h, respectively, in ZnCl2-treated C2-NEO cells(Fig. 8
, A
, II)
. In addition, the down-regulation of cyclinD1 and the accumulation of pRb (underphosphorylated) were acceleratedand occurred 2448 h earlier in ZnCl2-treated versus untreated(control) C2-NEO cells (Fig. 8
, B
and C
, III)
. Similarly,the reduction in c-myc expression characteristic for C2C12 differentiatingcells (Fig. 9I)
was detected in ZnCl2-treated clone 8 cellsat least 24 h earlier than in control cells (Fig. 9III)
. Itis thus concluded that PKR is involved in the regulation ofcell cycle-associated proteins.

Fig. 8. Determination of cell cycle-associated proteins. The levels of p21WAF1 (A), cyclin D1 (B), and pRb (C) were determined by immunoblot analysis of cell extracts prepared from cultures treated as indicated in the figure and described in Fig. 5.

Fig. 9. Determination of c-myc synthesis. The rate of synthesis of c-myc was determined by immunoblot analysis in extracts prepared from C2C12 (I), C2-NEO (II), and clone 8 (III) cells treated as indicated in the figure.

The IFN system as a whole has been previously shown to exhibitantiproliferation properties against a variety of cell types(34)
. Furthermore, it seems that molecular events associatedwith the cell cycle are some of the major targets affected byIFN. Thus, the reduction in c-myc expression, the accumulationof the underphosphorylated form of pRb (36, 37, 38, 39)
withthe concomitant down-regulation of cyclins and CDKs (40, 41,42)
, the reduced levels of the active E2F family of transcriptionfactors (38, 40, 43)
, and the induction of CDK-inhibitoryproteins (38, 41, 42)
were reported to be the result of IFNactivity. However, most of the IFN-induced biological activitiesare mediated by a variety of proteins activated by IFN via aunique signal transduction pathway (44)
. Accordingly, PKR hasbeen shown to mediate IFN-induced c-myc suppression in M1 myeloidleukemia cells (20)
. In addition, the ratio of the transcriptionalactivator of IFN-induced genes (IRF-1) to a suppressor of thesegenes (IRF-2) is high in growth-arrested cells and low in proliferatingcells. Furthermore, deletion of the IRF-1-encoding gene or overexpressionof IRF-2 may result in the development of tumors, includingthose of human origin (45)
. Finally, it has been demonstratedrecently that ectopic expression of 2-5A synthetase in myeloidcells induces cell growth arrest, a reduction in c-myc expression,an accumulation of the underphosphorylated form of pRb, andthe appearance of a myeloid differentiation marker (46)
.

In agreement with the concept that IFN-induced proteins playa role in the regulation of cell growth and differentiation,we show in the present report that PKR is activated during C2C12myogenic cell differentiation. These results support earlierfindings on the induction of PKR activity in rat primary skeletalmuscle cultures (28)
or in differentiating rat L8 myogeniccells (30)
. It is not surprising to see that, even in dividingC2C12 cells, an elevated level of PKR was observed at 96 h afterthe initiation of the experiment (Fig. 1)
because spontaneousdifferentiation is common in crowded cultures. This was accompaniedby cell growth arrest (Fig. 4
, A
and B)
and by elevated levelsof muscle-specific proteins (Fig. 5
, A
, I
and B
, I)
; Fig.7
) detected at late times in C2C12 cells cultivated in GM.However, the most striking phenomenon demonstrated in our studywas the fact that ectopic expression of PKR in myogenic cellsexposed to ZnCl2 induced a variety of morphological, biochemical,and molecular changes characteristic of myogenic differentiation.Thus, although complete elongated myotubes were not detectedin these cultures, a change in cell morphology and the formationof short myotubes consisting of three cells were common (Fig.3D)
. In addition, a retardation of cell growth (Fig. 4
, C
andD)
coupled with the accelerated appearance of muscle-specificproteins creatine kinase and troponin T (Fig. 5
, A
, II
andB
, II)
and myogenic transcription factors myoD and myogenin(Fig. 6B
, II
; Fig. 7
, A
, III
and B
, III
) was evident intransfected cells expressing PKR. Finally, an induction of theexpression of p21WAF1, accompanied by a reduction in the levelsof cyclin D1 and c-myc as well as an accumulation of the underphosphorylatedform of pRb, was also observed in these cells (Fig. 8
, ABC
,III
; Fig. 9
, III
). According to our view, PKR is most likelyinvolved in the down-regulation of gene expression, possiblyby the specific inhibition of the translation of certain mRNAmolecules. The induction of gene expression in myogenesis, onthe other hand, may then follow or be the result of an independentsignal transduction pathway and therefore is not directly relatedto PKR activity.

Recently, Datta et al.(31)
reported an additional IFN-inducedprotein, p202, whose level is increased during the differentiationof C2C12 cells. However, in contrast to the results obtainedin our report with PKR, ectopic expression of the gene encodingp202 in C2C12 cells inhibited rather than enhanced muscle differentiation.Thus, overexpression of p202 reduced the level of myoD and inhibitedthe transcriptional activation of both myoD and myogenin. Thediscrepancy between the observed elevated level of p202 duringdifferentiation and the inhibition of myoD and myogenin activationby ectopic expression of p202 is explained by Datta et al.(31)to be the result of early expression in the transfected cells.PKR, on the other hand, seems to be sufficient to induce anincrease in muscle-specific proteins. Therefore, it must beconcluded that p202 and PKR operate on two different levels,although both are activated during muscle differentiation.

An important finding in our study is the enhanced accumulationof the underphosphorylated form of pRb in PKR-expressing C2C12cells (Fig. 8C
, III)
. As mentioned above, this is accompaniedby a reduction in the level of both cyclin D1 and CDK4
andan increase in the synthesis of p21WAF1. pRb seems to be anessential component in the terminal differentiation of C2C12cells because expression of antisense Rb-1 RNA inhibits thisprocess (47)
. In addition, p21WAF1 is markedly induced duringmuscle differentiation (33, 48)
, indicating that pRb mustretain its active underphosphorylated form during terminal differentiation.Based on the data presented in our report, we conclude thatin C2C12 cells, PKR is sufficient to initiate a differentiationprocess characterized by cell growth arrest, the appearanceof muscle-specific proteins, changes in the level of cell growth-associatedfactors, and the partial fusion of myoblasts. Although the developmentof the embryo appears to be normal in PKR knockout mice (49)
,it is likely that a PKR homologue that has not yet been identifiedor other redundant proteins with similar functions (2-5A synthetase,for example) are activated in this case. This notion may notapply to committed myogenic cells, such as C2C12 cells becausetransfection of these cells with a dominant negative mutantof PKR leads to a significant inhibition of the myogenic process.5Thus, taken together, our data suggest that PKR is an importantelement in the regulation of myogenesis.

The cDNA fragment was excised from the vector by HindIII. Thisfragment was then subcloned in the HindIII site of BluescriptSK, resulting in two possible orientations. To distinguish betweenthe two constructs, several plasmid preparations were digestedwith SphI and XbaI. Ligation in the sense orientation was obtainedwhen the resulting fragments were 0.4- and 5-kb long. In thefinal stage, one of these plasmids (pBS-SK-PKR) was digestedwith SalI and XbaI, and the PKR-containing fragment was ligatedinto the polylinker SalI-XbaI site of plasmid pMSa (50)
. Thisplasmid contains the metallothionein promoter. Before the finalstep, an extra SalI site in pMSa was removed by SphI, followedby self-ligation. The final construct, pMPKR, was used in thisstudy.

Transfection
pMPKR was cotransfected with pSVneo (this plasmid contains theactive neomycin resistance gene fused to the early SV40 promoter;Ref. 51
) into C2C12 cells by electroporation. Approximately2 x 107 cells/ml were suspended in 250 µl of GM to which200 µl of sucrose buffer [272 mM sucrose and 7 mM Na3PO4(pH 7.4)] and 50 µl of DNA containing 15 µg of pMPKRand 1 µg of pSVneo were added. Electroporation was performedat 400 V and a capacitance of 500 µF using the Bio-Radgene pulsar II apparatus (Bio-Rad Laboratories, Hercules, CA).The cells were then transferred into 10-cm dishes containingDMEM supplemented with 20% FCS. After 48 h of incubation inGM, the cultures were subdivided at a ratio of 1:10, and G418(Calbiochem-Novabiochem Corp., La Jolla, CA) (800 µg/ml)was added 24 h later. After an additional 14 days, most of thecells died, and single colonies were visible. About 30 cloneswere removed by trypsin-EDTA solution, resuspended in GM withG418, and expanded. For a negative control, C2C12 cells weretransfected with 1 µg of pSVneo only. Eight clones weresimilarly isolated. A representative clone, C2-NEO, was usedthroughout this study.

Nuclear Extract.
Frozen pellets were prepared as described for total extracts,thawed in buffer W with 10 mM NaCl only, and centrifuged at10,000 xg for 20 min. The nuclear pellet was resuspended inoriginal buffer W and centrifuged again. The supernatant wascollected and kept in liquid nitrogen. These extracts were usedfor the determination of cyclin D1 by immunoblot analysis.

Protein Analysis by Immunoblotting
Total or nuclear cell extracts were prepared as described above.Samples (20 µg) were loaded on polyacrylamide-SDS geland analyzed by immunoblotting. We used the rainbow-coloredproteins as a molecular weight marker (Amersham International).

Determination of Specific RNA Transcripts
For each treatment, three 10-cm tissue culture dishes were used.Total RNA was extracted with Tri-reagent (Molecular ResearchCenter, Inc., Cincinnati, OH) according to the protocol suppliedby the manufacturer. Samples containing 30 µg of RNA wereanalyzed on 1% agarose gels in running buffer containing formaldehyde,followed by blotting onto nitrocellulose membrane filters (NitroPlus;MSI, Westboro, MA), as described previously (52)
. The ethidiumbromide-stained 18S and 28S bands of rRNA in each lane weredetected on both gels and filters by UV light. No significantdifferences in intensity between the lanes were observed. Forhybridization, the nitrocellulose filters were first prehybridizedat 42°C for 2 h in prehybridization buffer as describedby Sambrook et al.(52)
, with the addition of 0.1 mg/ml single-strandedsalmon sperm DNA. The labeled probe was then added at 12x 106 cpm/ml. Incubation was for 24 h at 42°C, and thenthe filters were washed once with 1 x saline-sodium phosphate-EDTA(15 mM NaH2PO4, 150 mM NaCl, and 1 mM EDTA) and 0.5% SDS for30 min at room temperature and once with 0.1 x saline-sodiumphosphate-EDTA and 0.1% SDS for 30 min at 50°C. Filterswere dried and exposed for autoradiography.

Determination of Growth Characterization
In the case of wild-type C2C12 cells, 1 x 105 cells/5-cm tissueculture plate were seeded in GM. One day later, the medium wasreplaced with either GM (for dividing cells) or DM (for differentiatingcells). This was considered zero time. With the transfectedclones, the cells were seeded at 1 x 105 cells/5-cm tissue cultureplate in GM, and some of the cultures were treated 3 h laterwith ZnCl2 (zero time). In all cases, the determination of thegrowth rate or thymidine incorporation was performed as describedbelow.

At the appropriate times, groups of three plates/point werecollected, the medium was removed, the plates were washed twicewith PBS, and the cells were collected with trypsin-EDTA, resuspendedin PBS, centrifuged, and resuspended again in 0.4% trypan bluein PBS (Sigma). Vital cells were counted in a hemocytometer,using five different fields/count. The SD of all the countsper time (total, 15 counts) was determined.

Thymidine Incorporation.
Cultures were prepared as described above. At the indicatedtimes, the medium was removed from the plate and replaced withfresh medium containing 1 µCi/ml [3H]thymidine (AmershamInternational) for 1.5 h. The cultures were then washed threetimes with cold PBS. The cells were lysed with 1% SDS for 10min at 37°C, and the lysates were moved to test tubes. Anequal volume of 20% trichloroacetic acid was added, and thetubes were kept on ice for 20 min. The samples were then filteredthrough Whatman 25 mm GF/c filters (supplied by Tamar, Ltd.,Jerusalem, Israel). The filters were dried, placed in toluene-basedscintillation fluid, and counted in Packard 1600 TR liquid analyzer.Each point represents the average of three different measurements.

Determination of Creatine Kinase Activity
Cultures were washed with Ca2+- and Mg2+-free PBS and homogenizedin 0.1 M sodium phosphate buffer (pH 7.0) supplemented with0.1% Triton X-100. The enzymatic activity was determined asdescribed by Shainberg et al.(53)
. The ATP formed by the interactionof ADP with creatine phosphate phosphorylates glucose in thepresence of hexokinase, yielding glucose-6 phosphate. The latterreduces NADP to NADPH, which is determined by recording theabsorption at 340 nm.

Footnotes

The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 Supported by grants from the Paula Better Estate, the HarvestCancer Fund, the Bar-Ilan University Research Authority, andthe Brazilian Friends of the Israel Cancer Association.